Bile acids, which aid in the absorption of lipids by the intestine and are required for the biliary elimination of compounds that cannot be excreted in the urine, exist primarily as glycine or taurine conjugates as the result of hepatic metabolism. It is known that this conjugation of bile acids is critical for their role in intestinal absorption but it is not known to what extent it is important for bile formation. We propose to examine this point using non-conjugable bile acids. We will also determine if there is enhanced conjugation of certain types of bile acids and what effect this might have on the solubility of cholesterol in bile. We will also investigate the regulatory factors affecting rates of conjugation. Theses studies will involve the indepth characterization of both the enzymology of the two enzymes of bile acid conjugation (cholyl-CoA synthetase and cholylCoA:Tau(Gly)N-acyltransferase), and studies of the basis for the diurnal variation in rates of conjugation. We will also follow up the indication that these enzymes can catalyze an esterification of bile acids with ethanol to form the insoluble and potentially toxic ethyl esters. Another aspect of conjugation that we have recently found interestng is the evolution of the mammalian Gly/Tau N-acyltransferases. These enzymes are 15,000 daltons smaller than the Tau:N-Acyltransferase from non-mammals. We will determine whether the mammalian and non-mammalian forms of the enzyme arose from a common ancestral gene by looking for regions of homology in their acid sequences We will determine if there are significant differences in active site chemistry between mammalian and non-mammalian forms. We will also try to explain the great variability in the specificity for glycine and taurine amongst the mammalian forms of the enzyme. Another important research problem has developed from our finding that physiologic concentrations of bile acid inhibit the detoxication of electrophilic Xenobiotics catalyzed by the glutathione S-transferases. This inhibition is non-competitive with substrates so we need to determine where the bile acid binding site is on the enzyme and the basis for the inhibition. We will determine if bile acids interfere with the ability of these enzymes to serve as transport proteins for non-polar Xenobiotics to prevent acetaminophen (etc.) hepatoxicity.